1. Field of the Invention
The present invention relates to a detection method, a detection apparatus, a sample cell for detection and a kit for detection to detect a substance to be detected (a detection target substance) in a sample.
2. Description of the Related Art
Conventionally, in the field of bio-measurement and the like, a fluorescence detection method is widely used as a highly accurate and easy measurement method. In the fluorescence detection method, a sample that is presumed to contain a detection target substance that outputs fluorescence by being excited by irradiation with light having a specific wavelength is irradiated with excitation light having the specific wavelength. At this time, fluorescence is detected to confirm the presence of the detection target substance. Further, when the detection target substance is not a phosphor (fluorescent substance), a substance that has been labeled with a fluorescent dye and that specifically binds to the detection target substance is placed in contact with the sample. Then, fluorescence from the fluorescent dye is detected in a manner similar to the aforementioned method, thereby confirming the presence of the bond between the detection target substance and the substance that specifically binds to the detection target substance. In other words, presence of the detection target substance is confirmed, and this method is widely used.
In bio-measurement, an assay is performed, for example, by using a sandwich method, a competition method or the like. In the sandwich method, when an antigen, as a detection target substance, contained in a sample needs to be detected, a primary antibody that specifically binds to the detection target substance is immobilized on a substrate (base), and a sample is supplied onto the substrate to make the detection target substance specifically bind to the primary antibody. Further, a secondary antibody to which a fluorescent label has been attached, and that specifically binds to the detection target substance, is added to make the secondary antibody bind to the detection target substance. Accordingly, a so-called sandwich structure of (primary antibody)-(detection target substance)-(secondary antibody) is formed, and fluorescence from the fluorescent label attached to the secondary antibody is detected. In the competition method, a competitive secondary antibody that competes with the detection target substance, and that specifically binds to a primary antibody, and to which a fluorescent label has been attached, binds to the primary antibody in such a manner to compete with the detection target substance. Further, fluorescence from the competitive secondary antibody that has bound to the primary antibody is detected.
When the assay is performed as described above, an evanescent fluorescence method has been proposed. In the evanescent fluorescence method, fluorescence is excited by evanescent light to detect fluorescence only from the secondary antibody that has bound, through the detection target substance, to the primary antibody immobilized on the substrate, or fluorescence only from the competitive secondary antibody that has directly bound to the primary antibody. In the evanescent fluorescence method, fluorescence excited by evanescent waves that extend from the surface of the substrate is detected. The evanescent waves are generated by making excitation light that totally reflects on the surface of the substrate enter the substrate from the back side of the substrate.
Japanese Unexamined Patent Publication No. 2005-077338 (Patent Literature 1) proposes an evanescent fluorescence method. In the evanescent fluorescence method disclosed in Patent Literature 1, instead of immobilizing the primary antibody on the substrate, a bound product (bound substance) of (primary reaction body)-(detection target substance)-(secondary reaction body) is formed in liquid phase. Further, the bound product is localized in an area to which the evanescent waves extend, and fluorescence from the bound product is detected. Specifically, the primary reaction body that includes a primary antibody and a magnetic material and the secondary reaction body that includes a fluorescent substance and the secondary antibody are bound to the detection target substance to obtain the bound product. The magnetic material contained in the primary reaction body is attracted by a magnet, and the bound product is localized.
Meanwhile, in the evanescent fluorescent method, methods using electric-field enhancement effects by plasmon resonance are proposed to improve the sensitivity of detection in U.S. Pat. No. 6,194,223 (Patent Literature 2), M. M. L. M Vareiro et al., “Surface Plasmon Fluorescence Measurements of Human Chorionic Gonadotrophin: Role of Antibody Orientation in Obtaining Enhanced Sensitivity and Limit of Detection”, Analytical Chemistry, Vol. 77, pp. 2426-2431, 2005 (Non-Patent Literature 1), and the like. In a surface plasmon enhancement fluorescence method, a metal layer is provided on the substrate, and excitation light is caused to enter the interface between the substrate and the metal layer from the back side of the substrate at an angle greater than or equal to a total reflection angle to generate surface plasmon resonance in the metal layer. Further, fluorescent signals are enhanced by the electric field enhancement action of the surface plasmons to improve the S/N (signal to noise) ratio.
Similarly, in the evanescent fluorescence method, a method using electric field enhancement effects by a waveguide mode is proposed in Spring 2007, the Japan Society of Applied Physics, Collection of Presentation Abstracts, No. 3, p. 1378 (Non-Patent Literature 2). In this optical waveguide mode enhanced fluorescence spectroscopy (OWF), a metal layer and an optical waveguide layer including a dielectric and the like are sequentially formed on the substrate. Further, excitation light is caused to enter the substrate from the back side of the substrate at an angle that is greater than or equal to the total reflection angle to induce an optical waveguide mode in the optical waveguide layer by irradiation with the excitation light. Further, fluorescent signals are enhanced by the electric field enhancement effect by the optical waveguide mode.
Further, Specification of U.S. Patent Application Publication No. 20050053974 (Patent Literature 3) and T. Liebermann and W. Knoll, “Surface-plasmon field-enhanced fluorescence spectroscopy”, Colloids and Surfaces A, Vol. 171, pp. 115-130, 2000 (Non-Patent Literature 3) propose a method for extracting radiation light (SPCE: Surface Plasmon-Coupled Emission) from the prism side. In the method, instead of detecting fluorescence output from a fluorescent label excited in the electric field enhanced by surface plasmons, the fluorescence newly induces surface plasmons in the metal layer, and radiation light by the newly induced plasmons is extracted from the prism side.
As described above, in bio-measurement or the like, various kinds of methods have been proposed as a method for detecting the detection target substance. In the methods, plasmon resonance or an optical waveguide mode is induced by irradiation with excitation light, and a fluorescent label is excited in an electric field enhanced by the plasmon resonance or the optical waveguide mode, and the fluorescence is directly or indirectly detected.
Further, in surface plasmon resonance measurement apparatuses, methods for increasing the concentration of detection target substance in a region on the sensor portion, the region to which evanescent waves extend from the sensor portion, are proposed in Japanese Unexamined Patent Publication No. 9 (1997)-257702 (Patent Literature 4), Japanese Unexamined Patent Publication No. 2007-085770 (Patent Literature 5), and the like. In Patent Literature 4, Patent Literature 5 and the like, voltage is applied to a sample to attract the detection target substance to the sensor portion, and measurement is performed. In these methods, the pH (potential of hydrogen) of a buffer solution is adjusted to adjust the charge state of a detection target substance, such as protein and nucleic acid. Further, voltage is applied in a state in which the detection target substance is positively or negatively electrified, thereby attracting the detection target substance to the sensor portion.
The method for localizing the detection target substance by application of voltage can achieve a certain effect. Further, Patent Literature 4 describes that in surface plasmon resonance measurement apparatuses, when the detection target substance is attracted to a region within approximately 100 nm from the sensor portion, which the evanescent waves reach, it is possible to reduce the variation in signals.
However, since both of the size and the charge of the detection target substance are small, the attraction effect by application of voltage is weaker than Brown motion of the detection target substance. Therefore, it is difficult to efficiently attract the detection target substance to the surface of the sensor portion. Further, it is necessary to provide a means for applying voltage to the liquid sample. Therefore, there is a problem that the structure of the apparatus becomes complicated.
Further, the electric field enhancement effects by surface plasmon resonance and optical guide mode sharply attenuate as a distance from the surface of the metal layer or the optical waveguide layer increases. Therefore, there is a problem that when the distances from the surface to the fluorescent labels even slightly change, signals from the fluorescent labels become different from each other, and varied. Hence, it is necessary to attract the fluorescent labels within a range of approximately 50 nm from the surface.
For example, FIG. 20 is a schematic diagram illustrating an apparatus for detecting fluorescence by an electric field enhancement effect by surface plasmon resonance. In FIG. 20, the vicinity of a sensor portion of the apparatus is illustrated. A gold film (thin-film, coating or layer) 102 is deposited on a surface of a prism (substrate) 101. Further, primary antibody B1 is immobilized on the gold film 102. When a sandwich assay is performed, fluorescence from a fluorescent label (fluorescent dye molecule f in this case) attached to labeling secondary antibody B2 is detected. The labeling secondary antibody B2 binds to the primary antibody B1 through antigen A. Excitation light is caused to enter the interface between the prism 101 and the gold film 102 at an angle greater than or equal to the total reflection angle to excite surface plasmons on the surface of the gold film 102. Accordingly, the electric field on the surface of the gold film 102 is enhanced. The fluorescent label (fluorescent dye molecule) f is excited in the enhanced electric field, and fluorescence is output. In FIG. 20, the graph shows distance-dependent characteristic of the strength (magnitude) of the electric field, the distance being measured from the surface of the sensor portion (surface of the gold film). As the graph shows, the strength of the electric field sharply decreases as the distance from the surface increases.
At this time, the maximum distance from the surface of the sensor portion to the fluorescent label f of the labeling secondary antibody is approximately 50 nm. When the distance from the surface of the sensor portion is approximately 50 nm, the intensity of fluorescence attenuates by 30% or more. Further, the primary antibody B1 is not always immobilized upright on the surface of the sensor portion, and the primary antibody B1 may fall along the surface by the flow of liquid, a three-dimensional obstacle or the like, and be immobilized in a lying or inclined state. Consequently, the distance from the surface of the fluorescent label f to the surface of the sensor portion is varied, and the intensity of the signal is varied.